The present invention relates to a multiprotein surveillance complex comprising human Upf1p eucaryotic Release Factor 1 and eucaryotic Release Factor 3 which is involved in modulation of the efficiency of translation termination and degradation of aberrant mRNA. Identification of this complex provides an in vitro assay system for identifying agents that: affect the functional activity of mRNAs by altering frameshift frequency; permit monitoring of a termination event; promote degradation of aberrant transcripts; provide modulators (inhibitors/stimulators) of peptidyl transferase activity during initiation, elongation, termination and mRNA degradation of translation. Such agents which may be antagonists or agonists, are useful for screening, and diagnostic purposes, and as therapeutics for diseases or conditions which are a result of, or cause, premature translation.
Recent studies have demonstrated that cells have evolved elaborate mechanisms to rid themselves of aberrant proteins and transcripts that can dominantly interfere with their normal functioning (reviewed in Gottesman et al. 1997, He et al. 1993, Jacobson and Peltz 1996, Ruiz-Echevarria et al. 1996, Suzuki et al. 1997, Weng et al. 1997, Maquat, 1995, Pulak and Anderson, 1993). Such pathways can be viewed both as regulators of gene expression and as sensors for inappropriate polypeptide synthesis. The nonsense-mediated mRNA decay pathway (NMD) is an example of a translation termination surveillance pathway, since it eliminates aberrant mRNAs that contain nonsense mutations within the protein coding region (Gottesman et al. 1997, He et al. 1993, Jacobson and Peltz, 1996, Ruiz-Echevarria et al. 1996, Suzuki et al. 1997, Weng et al. 1997, Pulak and Anderson, 1993, Caponigro and Parker, 1996, Maquat, 1995). The NMD pathway has been observed to function in all eucaryotic systems examined so far and appears to have evolved to ensure that termination of translation occurs at the appropriate codon within the transcript. Transcripts containing premature nonsense codons are rapidly degraded, thus preventing synthesis of incomplete and potentially deleterious proteins. There are well over two hundred genetic disorders which can result from premature translation termination (McKusick, 1994).
The proteins involved in promoting NMD have been investigated in C. elegans mammalian cells and in the yeast Saccharomyces cerevisiae. Three factors involved in NMD have been identified in yeast. Mutations in the UPF1, UPF2, and UPF3 genes were shown to selectively stabilize mRNAs containing early nonsense mutations without affecting the decay rate of most wild-type mRNAs (He and Jacobson 1995, Lee and Culbertson 1995, Leeds et al. 1992, Leeds et al. 1991, Cui et al. 1995). Recent results indicate that the Upf1p, Upf2p and Upf3p interact and form a complex (He and Jacobson 1995, He et al. 1997, Weng et al. 1996b). In C. elegans, seven smg alleles have been identified which result in an increased abundance of nonsense-containing transcripts (Pulak and Anderson, 1993). A human homologue of the UPF1 gene, called RENT1 or HUPF 1, has been identified, indicating that NMD is an evolutionarily conserved pathway (Perlick et al. 1996, Applequist et al. 1997).
Although the cellular compartment in which NMD occurs in mammalian cells is controversial (Weng et al., 1997; Maquat, 1995; Zhang and Maquat 1997), it appears that in yeast, however, NMD occurs in the cytoplasm when the transcript is associated with ribosomes. Results supporting this conclusion are the following; 1) nonsense-containing and intron-containing RNAs that are substrates of the NMD pathway in yeast become polysome-associated and are stabilized in the presence of the translation elongation inhibitor cycloheximide (Zhang et al. 1997). The polysome associated RNAs, however, regain their normal rapid decay kinetics when the drug is washed out of the growth medium and translation resumes (Zhang et al., 1997); 2) Upf1p, Upf2p and Upf3p have been shown to be associated with polysomes (Peltz et al., 1993a, 1994; Atkin et al., 1995; Atkin et al., 1997); 3) as revealed by fluorescent in situ hybridization analysis, the cytoplasmic abundance of an intron-containing LacZ reporter RNA containing mutations in the 5xe2x80x2 splice site or branch point was dramatically reduced in UPF1+ strain but increased in cytoplasmic abundance in upf1xcex94 cells (Long et al., 1995); 4) NMD can be prevented by nonsense-suppressing tRNAs (Losson and Lacroute, 1979; Gozalbo and Hohmann, 1990; Belgrader et al., 1993); 5) the NMD pathway is functional only after at least one translation initiation/termination cycle has been completed (Ruiz-Echevarria and Peltz, 1996; Ruiz-Echevarria et al., 1998; Zhang and Maquat, 1997). Furthermore, a translation reinitiation event can prevent activation of the NMD pathway (Ruiz-Echevarria and Peltz, 1996; Ruiz-Echevarria et al., 1998; Zhang and Maquat, 1997). Taken together, these results indicate that the NMD pathway in yeast is a cytoplasmic and translation-dependent event. The rent1/hupf1 protein is also predominantly cytoplasmic (Applequist et al. 1997)
The yeast UPF1 gene and its protein product have been the most extensively investigated factor of the putative surveillance complex (Czaplinski et al. 1995, Weng et al. 1996a,b, Weng et al., 1998, Altamura et al. 1992, Cui et al. 1996, Koonin, 1992, Leeds et al. 1992, Atkin et al. 1995,1997). The Upf1p contains a cysteine- and histidine-rich region near its amino terminus and all the motifs required to be a member of the superfamily group I helicases. The yeast Upf1p has been purified and demonstrates RNA binding and RNA-dependent ATPase and RNA helicase activities (Czaplinski et al. 1995, Weng et al. 1996a,b). Disruption of the UPF1 gene results in stabilization of nonsense-containing mRNAs and suppression of certain nonsense alleles (Leeds et al. 1991, Cui et al. 1995, Czaplinski et al. 1995, Weng et al. 1996a; Weng et al. 1996b).
The ability to modulate translation termination has important implications for treating diseases associated with nonsense mutations. As with any biological system, there will be a small amount of suppression of a nonsense mutation, resulting in expression of a full length protein (which may or may not include an amino acid substitution or deletion). In the natural state, such low quantities of full length protein are produced that pathology results. However, by stabilizing the nonsense mRNA, the likelihood of xe2x80x9cread-throughxe2x80x9d transcripts is dramatically increased, and may allow for enough expression of the protein to overcome the pathological phenotype.
The nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations. The product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs. The results presented here demonstrate that the yeast and human forms of the Upf1p interact with both eucaryotic translation termination factors eRF1 and eRF3. Consistent with Upf1p interacting with the eRFs, the Upf1p is found in the prion-like aggregates that contain eRF1 and eRF3 observed in yeast [PSI+] strains. These results indicate that interaction of the Upf1p with the peptidyl release factors is a key event in the assembly of the putative surveillance complex that enhances translation termination monitors whether termination has occurred prematurely and promotes degradation aberrant transcripts.
This invention provides an isolated complex comprising a human Upf1p protein, a peptidyl eucaryotic release factor 1 (eRF1) and a peptidyl eucaryotic release factor 3 (eRF3), wherein the complex is effective to modulate peptidyl transferase activity. In one embodiment, this invention further comprises a human Upf3p and Upf2p.
This invention provides an agent which binds to the complex comprising an amount of a human Upf1p protein, a peptidyl eucaryotic release factor 1 (eRF 1) and a peptidyl eucaryotic release factor 3 (eRF3) effective to modulate translation termination. This invention provides an agent which binds to the complex of claim 1, wherein the agent inhibits ATPase of Upf1p; GTPase activity of eRF1 or eRF3; or RNA binding to a ribosome. This invention provides an agent which inhibits or modulates the binding of human Upf1p to eRF 1, or eRF3 or eRF1 or eRF3 to Upf1p. This invention provides an agent which inhibits or modulates the binding of human Upf3p to eRF1, or eRF3 or eRF1 or eRF3 to Upf3p. This invention provides an agent which facilitates the binding of human Upf1p to eRF1 or eRF3; or eRF3 or eRF1 or eRF3 to Upf1p. This invention provides an agent which facilitates the binding of human Upf3p to eRF1 or eRF3; or eRF3 or eRF1 or eRF3 to Upf1p. This invention provides an agent which modulates the binding of human Upf1p, eRF1 or eRF3 to a ribosome.
This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the complex in an amount effective to facilitate translation termination, thereby modulating the peptidyl transferase activity.
This invention provides a method of modulating peptidyl transferase activity during translation, comprising contacting a cell with the agent, in an amount effective to suppress non-sense translation termination, thereby modulating the peptidyl transferase activity. The peptidyl transferase activity during translation occurs during initiation, elongation, termination and degradation of mRNA.
This invention provides a method of modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts, comprising contacting a cell with the agent, in an amount effective to inhibit the binding of human Upf1p to eRF1, or eRF3; or eRF1 or eRF3 to Upf1, thereby modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts.
This invention provides a method of modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts, comprising contacting a cell with an agent, which inhibits the ATPase/helicase activity of Upf1p; the GTPase activity of eRF1 or eRF3, or binding of RNA to a ribosome, thereby modulating the efficiency of translation termination of mRNA at a non-sense codon and/or promoting degradation of abberant transcripts.
This invention provides a method of screening for a drug involved in peptidyl transferase activity during translation comprising: a) contacting cells with a candidate drug; and b) assaying for modulation of the complex, wherein a drug that modulates the complex is involved in peptidyl transferase activity or enhancing translation termination.
This invention provides a method of screening for a drug involved in enhancing translation termination comprising: a) incubating the drug and the complex; and b) measuring the effect on non-sense suppression, thereby screening for a drug involved in enhancing translation termination. The assays may be a RNA or NTPase assays, such as ATPase or GTPase.
This invention provides a method of modulating the efficiency of translation termination of mRNA and/or degradation of abberant transcripts in a cell, said method comprising: a) providing a cell containing a vector comprising the nucleic acid encoding proteins of the complex, the complex; or an antisense molecule thereof; b) overexpressing said nucleic acid in said cell to produce an overexpressed complex so as to interfere with the function of the complex.
This invention provides method for identifying a disease state involving a defect in the complex comprising: (a) transfecting a cell with a nucleic acid which encodes the complex; (b) determining the proportion of the defective complex of the cell after transfection; (c) comparing the proportion of the defective complex of the cell after transfection with the proportion of defective complex of the cell before transfection.
This invention provides a method for treating a disease associated with peptidyl transferase activity, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the complex or the agents, and a pharmaceutical carrier or diluent, thereby treating the subject.